Assessment of microbiota present on a Portuguese historical stone convent using high‐throughput sequencing approaches

Abstract The study performed on the stone materials from the Convent of Christ revealed the presence of a complex microbial ecosystem, emphasizing the determinant role of microorganisms on the biodecay of this built cultural heritage. In this case study, the presence of Rubrobacter sp., Arthrobacter sp., Roseomonas sp., and Marinobacter sp. seems to be responsible for colored stains and biofilm formation while Ulocladium sp., Cladosporium sp., and Dirina sp. may be related to structural damages. The implementation of high‐throughput sequencing approaches on the Convent of Christ's biodecay assessment allowed us to explore, compare, and characterize the microbial communities, overcoming the limitations of culture‐dependent techniques, which only identify the cultivable population. The application of these different tools and insights gave us a panoramic view of the microbiota thriving on the Convent of Christ and signalize the main biodeteriogenic agents acting on the biodecay of stone materials. This finding highlighted the importance of performing metagenomic studies due to the improvements and the reduced amount of sample DNA needed, promoting a deeper and more detailed knowledge of the microbiota present on these dynamic repositories that support microbial life. This will further enable us to perform prospective studies in quarry and applied stone context, monitoring biogenic and nonbiogenic agents, and also to define long‐term mitigation strategies to prevent biodegradation/biodeterioration processes.

microorganisms can be cultivated by standard techniques and the cultivable microorganisms are under-represented considering the microbial diversity present in the Earth (González & Saiz-Jiménez, 2005;Sanchez-Moral et al., 2005). Despite the limitations inherent to this approach, culture-based techniques and the development of new culture media are still encouraged due to the advantage of having pure cultures isolated to carry out physiological and metabolic studies (Dakal & Arora, 2012;Dyda et al., 2018).
Nevertheless, to understand the phenomena that promote the degradation of cultural assets it is crucial to obtain a deeper knowledge about the colonizing microbial population. In this way, techniques based on nucleic acids allow the differentiation of microorganisms within complex microbial communities or the identification of isolated microorganisms (Portillo & Gonzalez, 2009). Therefore, DNA sequencing approaches are very useful to phylogenetic identification and have been applied in several areas, being suitable in artworks to analyze the microbial diversity (Cappitelli et al., 2009;Carmona et al., 2006;Olivares et al., 2013;Saarela et al., 2004;Schabereiter-Gurtner, Piñar, Vybiral, Lubitz, & Rolleke, 2001).
Small subunit ribosomal DNA genes like 16S and 18S universally present in all prokaryotes and eukaryotes, respectively, provide an efficient mean to identify microorganisms. These ribosomal sequences possess variable and highly conserved regions, which are used as phylogenetic markers to identify and distinguish the different microorganisms on all phylogenetic levels (Dakal & Arora, 2012;Kennedy & Clipson, 2003;Li, Zhang, He, & Yang, 2016;Zhang, Ping, Zhou, Wang, & Zhang, 2018). The internal transcribed spacer (ITS) region is also an effective tool for the identification of eukaryotic population (Anderson & Cairney, 2004;Dakal & Arora, 2012;Kennedy & Clipson, 2003). In the case of yeast, the sequencing of the D1/D2 domain of 26S/28S rDNA region has been used to identify these microorganisms from different sources. This approach is rapid and precise when compared with the physiological method for the yeast identification and has also been applied to study the phylogeny of different yeast groups and species-level differentiation (Dagar, Kumar, Mudgil, Singh, & Puniya, 2011;Hesham, Wambui, Ogola, & Maina, 2014;Kiyuna et al., 2012;Lv, Huang, Zhang, Rao, & Ni, 2013;Selbmann et al., 2014). Other nucleic acid approaches can be applied to detect uncultivable microorganisms. Recent developments in sequencing chemistries, bioinformatics, and automated instruments have revolutionized the knowledge of microbial diversity (Dyda et al., 2018;Edgar, Haas, Clemente, Quince, & Knight, 2011;England & Pettersson, 2005;Li et al., 2016;Mardis, 2008;Schubert, Lindgreen, & Orlando, 2016;Shendure & Ji, 2008;Zhang, Chiodini, Badr, & Zhang, 2011;Zhang et al., 2018;Zhou et al., 2015). The ongoing advances in genomics and sequencing technologies are allowing a new era of microbial community analyses using culture-independent approaches such as metagenomics, metaproteomics, metatranscriptomics, and proteogenomics which are fundamental for the full identification of the microbial diversity and to understand their interactions with biotic and abiotic factors (Rastogi & Sani, 2011).
This work aims to encompass a comprehensive multidisciplinary approach to characterize the biological colonization and consequently biodeterioration/biodegradation of an architectural stone material (Ançã limestone) present in the Convent of Christ, a UNESCO World Heritage monument located in Tomar (Central Portugal). This emblematic Convent has been studied by our research group since 2015 (Rosado et al., 2016) owing to the visible evidence of alteration on the stone materials which are responsible for structural and aesthetic damages like stains, biofilm formation, delamination, and detachment of stone fragments.
In order to reach an answer for the degradation/deterioration processes that are affecting the stone, culture-dependent methods (CDM), scanning electron microscopy (SEM), and HTS were applied in the multiple zones of the Convent showing different alteration processes to (a) characterize the microorganisms that are colonizing the stone surfaces and (b) evaluate the distribution patterns of the microbiota.

| Sampling process
The monumental complex of the Convent of Christ in Tomar (N39°36′14.8536″, W8°25′9.231″) is classified as UNESCO World Heritage monument, and it is an emblematic exemplar of the Portuguese history that possesses a wide diversity of sculpted stones with visible marks of biocontamination. This monument stands at the top of a hill in Tomar, a city with hot and dry summers, as well as cold winters, with precipitation and partially overcast sky.
Temperatures often reach 38°C in the summer and −1°C in the winter. The yearly rainfall average is 773 mm.
The sampling process was performed on representative areas of the stone materials present in the Convent of Christ, including the areas with evidence of alterations, under the coordination of a conservator-restorer ( Figure 1).
Microinvasive (chisel and scalpels) and noninvasive (swabs) methods were applied during the samples' collection, under semi-aseptic conditions (collection performed with sterile material but in outdoor environment). Microsamples of stone (10 samples with less than 1 mm 2 ) were removed using a small chisel to perform the material characterization, close to losses or cracks in order to avoid further damage. For the microbiological assays, samples were also collected under semi-aseptic conditions with sterile swabs and placed in a suspension of transport MRD medium (Maximum Recovery Diluent) until utilization.

| Biocolonization assessment
To assess the deterioration degree of the support and the presence of biocolonization, microfragments of stone were used as such or coated with Au-Pd (Balzers Union SCD 030) during 30 s, and observed in a HITACHI S-3700N variable pressure scanning electron microscope (VP-SEM) with an accelerating voltage of 10 kV. Microanalyses of the selected samples were performed using the same microscope coupled with a Bruker XFlash 5010 energy-dispersive X-ray spectrometer (EDX) to allow a microstructural characterization of the stones and to obtain the elemental composition (point analyses and 2D mapping). EDX analyses were performed at 20 kV.

| Cultivable population
The samples collected for biological assays were mechanically shaken during 24 hr at 150 rpm. After this period, each sample was inoculated in solid cultures containing different culture media (Nutrient Agar-NA, Malt Extract Agar-MEA, Cooke Rose Bengal-CRB, ASM-1 and BG-11), selective for several kinds of microorganisms (bacteria, fungi, and cyanobacteria, respectively). Bacterial isolation procedures were carried out in Nutrient Agar, at 30°C and for 48 hr. For fungal colonies' isolation, two standard mycological media (MEA and CRB) were used, and the cultures were incubated for 7 days at 28°C. For cyanobacterial development, the samples were inoculated in ASM-1 and BG-11 (solid and liquid media) and were incubated at room temperature (20-25°C) with periods of light exposure (16:8 hr light-dark cycle) for 1 month. After this period, the plates were maintained at the same temperature mentioned above to detect slow bacterial/fungal development.
The distinct single colonies obtained were subcultured onto NA/ MEA Petri dishes and maintained on NA/MEA slants at 4°C.
The different microbiological strains were identified following the standard methods (Domsch, Gams, & Anderson, 1980), based on their macro-and micromorphological characteristics. For each bacterial/fungal isolate, the genes encoding the 16S/ITS sequence, respectively, were amplified by PCR, purified, and sequenced by outsourcing service (STAB VIDA). The nucleotide sequences were aligned with those retrieved from the GenBank (NCBI) databases for the homology analysis using the BLASTN 2.8.0 program.  at Genoinseq. They were multiplexed automatically by the Miseq ® sequencer using the CASAVA package (Illumina) and quality-filtered with PRINSEQ software (Schmieder & Edwards, 2011) using the following parameters: (a) bases with average quality lower than Q25 in a window of five bases were trimmed, and (b) reads with less than 220 bases were discarded for V3-V4 samples and less than 100 bases for ITS2 samples. The forward and reverse reads were then merged by overlapping paired-end reads using the Adapter Removal v2.1.5 (Schubert et al., 2016) software with default parameters. The QIIME package v1.8.0 (Caporaso et al., 2010) was used for operational taxonomic unit (OTU) generation, taxonomic identification, sample diversity, and richness indexes' calculation. Sample IDs were assigned to the merged reads and converted to fasta format (split_li-braries_fastq.py, QIIME). Chimeric merged reads were detected and removed using UCHIME (Edgar et al., 2011)

| RE SULTS AND D ISCUSS I ON
Preliminary studies performed on the built stone areas of the Convent of Christ revealed evident signals of biocontamination linked to some weathering effects, which is leading the stone to serious problems (Rosado et al., 2016). Alteration products like calcium oxalates and carotenoids were identified on deteriorated areas of the stone, which seems to be correlated with the presence of microbial contamination as a result of their metabolic activity (Rosado et al., 2016).
Thus, to be able to answer the key questions of the stone alteration processes, complementary methodologies based on culture-dependent techniques, metagenomic approaches, and analytical methods were used to characterize the biocolonizers present on the stone building materials of the Convent of Christ. There are several advantages on the use of a multianalytical approach, which will be further presented and discussed in this paper.
As mentioned above, this monument exhibits formation of biofilms, stains, and structural degradation covering several outdoor areas ( Figure 1). The surrounding environment (temperature, humidity, sun, and wind) together with the bioreceptivity of the stone materials seems to contribute to its biocolonization (  present in complex systems like cultural heritage assets (Chimienti et al., 2016;Cutler et al., 2013;Dias et al., 2018;Rosado et al., 2014;Tonon et al., 2019). In this work, prokaryote and eukaryote communities were assessed by NGS.
The microbiota thriving on these altered stone areas is quite lichen is widespread throughout many areas of Europe, probably due to its reproductive strategy, its ability to exploit man-made substrata, and the absence of other competing species resulting from the increasing air pollution (Coutinho et al., 2013;Edwards, 2007;Pinna, 2014;Seaward, 1997;Tonon et al., 2019;Zagari, Antonelli, & Urzi, 2000).
Thus, advances in high-throughput analysis have provided new tools for the identification of microorganisms present in complex systems, which can help in the studies of cultural heritage field in order to define strategies to protect the assets from biocolonization and develop approaches to safeguard the monuments, being crucial the implementation of NGS technology in CHs workflows to fully characterize the microbiota.
Despite the fact that metagenomic analyses provide information about the genomic content of microorganisms (living or not), other approaches like metatranscriptomics and metaproteomics can be used to gain information on the active portion of the microbiota that thrive on stone monuments.
The whole perception of the biocolonizers and their effects enables the development of better intervention approaches to conduct the preservation of stone structures, prevent the loss of their aesthetic and historical value and structural integrity, benefiting both cultural and economic aspects (Aalil et al., 2016).
A long-term monitoring is ongoing in the Convent of Christ, encompassing in situ measurements of temperature and relative humidity, microanalytical analyses to detect additional alterations and assessment of biocolonizers changes, according to the yearly seasons.
Cleaning and preventing actions based on natural biocompounds, whose efficacy and effectiveness were already tested by our research group, will be considered to eliminate, control, and prevent microorganisms' development (Rosado et al., 2019) in the near future. Moreover, a screening of the microbial communities before and after the application of biocides as well as their monitoring throughout time is clearly needed, and this should be carried out in the framework of cultural heritage safeguard.

| CON CLUS IONS
The Convent of Christ is a great example of an important stone building that has suffered significant alterations apparently caused by biocolonization, providing an ecological niche for the survival of microorganisms like bacteria, yeast, fungi, and lichens.
The aesthetic damages throughout the stone of the Convent, such as stains and biofilms, seem to be induced by bacteria, mainly by the strains Rubrobacter sp., Arthrobacter sp., Roseomonas sp., and Marinobacter sp, while fungi (Ulocladium sp., Cladosporium sp.) and lichens (Dirina sp.) appear to be the main responsible for structural alterations.
Among the techniques (CDM, SEM, NGS) used for monitoring the microbiological agents that cause biodeterioration of cultural heritage assets, NGS is currently the most promising approach. With this innovative application, we hope to contribute, as much as possible, to the knowledge of the microbial population that are colonizing stone materials as well as to understand its effect on stone decay.
We believe that this study is a step forward for the implementation of an effective and accurate plan of conservation and intervention (including mitigation treatment) of a UNESCO World Heritage monument, the Convent of Christ, to avoid the destruction of this cultural asset and to promote its preservation and safeguard.

ACK N OWLED G M ENTS
The

E TH I C S S TATEM ENT
None required.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analyzed during this study are provided in full in the results section of this paper. Raw data sets from NGS results are available in Appendix.